Mitochondrial fluorescent probe independent of mitochondrial membrane potential as well as preparation method and application of mitochondrial fluorescent probe

Abstract

The invention discloses a mitochondrial fluorescent probe independent of mitochondrial membrane potential and a preparation method and application thereof, and the mitochondrial fluorescent probe independent of mitochondrial membrane potential provided by the invention can target and image mitochondria when the mitochondrial membrane potential is reduced and disappears.

Description

Technical Field

[0001] The present invention relates to the field of probes, and in particular to a mitochondrial fluorescent probe independent of mitochondrial membrane potential, its preparation method and application. Background Art

[0002] Mitochondria are organelles in cells that produce energy and are the main sites for aerobic respiration in cells. In addition to supplying energy to cells, mitochondria also participate in life processes such as cell differentiation, cell information transmission, and apoptosis, and have the ability to regulate cell growth and the cell cycle. Many diseases are closely related to the morphology, quantity, etc. of mitochondria. Mitochondrial fluorescence imaging is an important means for mitochondrial research, so mitochondrial fluorescent probes are widely used in medical, life science research and clinical diagnosis.

[0003] The existing mitochondrial fluorescent probes currently available are driven by the high mitochondrial membrane potential of living cells and aggregate in mitochondria, and can only be used for mitochondrial staining imaging in living cells, and cannot specifically stain biological samples with reduced or even lost mitochondrial membrane potential, including mitochondria in fixed cells. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a mitochondrial fluorescent probe, its preparation method and application to solve the problem that the existing mitochondrial fluorescent probes rely on high mitochondrial membrane potential.

[0005] According to a first aspect embodiment of the present invention, a mitochondrial fluorescent probe independent of mitochondrial membrane potential, the

[0006] mitochondrial fluorescent probe is a compound having a structure shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0007]

[0008] wherein, the R 1 includes any one of hydrogen or an alkyl group of C1-C4; the R 2 includes any one of hydrogen, an alkyl group of C1-C4 and an alkoxy group of C1-C4; the X includes a halogen atom, BF 4 and ClO 4 any one of them.

[0009] According to some embodiments of the present invention, the halogen atom is selected from any one of iodine, bromine, and chlorine.

[0010] According to some embodiments of the present invention, in the R 1 , the alkyl group of C1-C4 includes any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0011] According to some embodiments of the present invention, the R 2 Among them, the C1-C4 alkyl group includes any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0012] According to some embodiments of the present invention, the R 2 Among them, the C1-C4 alkoxy group includes any one of methoxy, ethoxy, propoxy and butoxy.

[0013] According to some preferred embodiments of the present invention, the mitochondrial fluorescent probe includes (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide.

[0014] In the present invention, R 1 is selected from hydrogen; R 2 is selected from methoxy; when the X is selected from iodine, the obtained mitochondrial fluorescent probe independent of mitochondrial membrane potential is (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-n-hexylquinolinium iodide.

[0015] According to a preparation method of the mitochondrial fluorescent probe according to the second aspect embodiment of the present invention, the preparation method includes the following steps:

[0016] S1. 4-Methylquinoline (Formula II) and halohexane (Formula III) are placed in a solvent and refluxed to react to generate 1-n-hexyl-4-methylquinolinium salt (Formula IV);

[0017] S2. 1-n-Hexyl-4-methylquinolinium salt, indole-3-carbaldehyde (Formula V) and a catalyst are mixed, refluxed and then the impurities are removed;

[0018] The catalyst includes piperidine.

[0019] According to some embodiments of the present invention, the solvent includes ethanol.

[0020] According to some embodiments of the present invention, the molar ratio of indole-3-carbaldehyde to 4-methylquinoline is 1:(1.0-2.0).

[0021] According to some embodiments of the present invention, in step S2, the reflux reaction time is 6h-24h.

[0022]

[0023] According to some preferred embodiments of the present invention, the indole-3-carbaldehyde is selected from 5-methoxy-3-formylindole; the halohexane is selected from iodohexane. Using 5-methoxy-3-formylindole and iodohexane as reactants, the preparation method of the mitochondrial fluorescent probe is as follows:

[0024] S01. Prepare an ethanol mixed solution of 4-methylquinoline and iodohexane;

[0025] S02. Heat and stir under reflux for three days;

[0026] S03. Add an ethanol solution of 5-methoxy-3-formylindole;

[0027] S04. Add the catalyst piperidine to the ethanol mixed solution, heat the ethanol mixed solution added with piperidine under reflux at 85 °C for one day, distill off the excess solvent, and slowly cool to room temperature to obtain the organic solid product to be purified;

[0028] S05. Purify the organic solid product to be purified by column chromatography, use dichloromethane / methanol as the eluent, and dry to obtain a purple-red powder. The purple-red powder is the mitochondrial fluorescent probe (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-n-hexylquinolinium iodide that does not depend on the mitochondrial membrane potential.

[0029] Use of a mitochondrial fluorescent probe according to an embodiment of the third aspect of the present invention in any one of A1) to A4):

[0030] A1) Labeling and / or localizing mitochondria for non-disease treatment diagnosis;

[0031] A2) Monitoring the related life activities of mitochondria for non-disease treatment diagnosis;

[0032] A3) Preparing a product for labeling and / or localizing mitochondria;

[0033] A4) Preparing a product for monitoring the related life activities of mitochondria.

[0034] According to some embodiments of the present invention, the mitochondria include mitochondria in cells with reduced mitochondrial membrane potential such as fixed cells.

[0035] Use of a mitochondrial fluorescent probe according to an embodiment of the fourth aspect of the present invention in preparing a product targeting mitochondria.

[0036] Use of a mitochondrial fluorescent probe according to an embodiment of the fifth aspect of the present invention in preparing a product for mitochondrial imaging.

[0037] The mitochondrial fluorescent probe provided by the present invention that does not rely on mitochondrial membrane potential realizes the application of a mitochondrial fluorescent probe that does not rely on mitochondrial membrane potential in imaging mitochondria in cells, especially in imaging the morphology, quantity, and distribution of mitochondria in biological samples with reduced or even lost mitochondrial membrane potential, including fixed cells.

[0038] The above-mentioned indoloquinoline hexyl salt fluorescent probes in the present invention are a new type of mitochondrial-specific recognition fluorescent probe molecules in cells. Compared with the existing mitochondrial fluorescent probes, the unique feature of the probes described in the present invention is that they do not rely on a high mitochondrial membrane potential and can image the morphology, quantity, and distribution of mitochondria in biological samples with reduced or even lost mitochondrial membrane potential, including fixed cells. They do not fluoresce in water / PBS / culture medium (without serum and antibiotics) solutions by themselves, but emit red fluorescence after binding to mitochondria in cells; at the same time, they have good membrane permeability and good compatibility for counterstaining.

[0039] The mitochondrial fluorescent probe provided by the present invention that does not rely on mitochondrial membrane potential can be used as a fluorescent probe to label the morphology, quantity, and distribution of mitochondria in cells, and can provide a simple and intuitive biological detection reagent for mitochondrial-related physiological and pathological research and clinical diagnosis. It has a wide range of applications and good effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of the embodiments in conjunction with the accompanying drawings, where:

[0041] Figure 1 is a confocal fluorescence micrograph of the co-staining of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide with mitochondrial deep red fluorescent probe (MitoTracker Deep Red) and mitochondrial green fluorescent probe (MitoTracker Green) on cancer cell HeLa cells.

[0042] Figure 2 is a confocal fluorescence micrograph of the co-staining of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide with mitochondrial deep red fluorescent probe (MitoTracker Deep Red) and mitochondrial green fluorescent probe (MitoTracker Green) on HeLa cells treated with CCCP.

[0043] Figure 3Confocal fluorescence micrograph of co - staining of (E)-4-(2-(5 - methoxy - 1H - indol - 3 - yl)vinyl)-1 - n - hexylquinolinium iodide and mitochondrial deep red fluorescent probe (MitoTracker Deep Red) on fixed normal cells HEK293.

[0044] Figure 4 Confocal fluorescence micrograph of staining of (E)-4-(2-(5 - methoxy - 1H - indol - 3 - yl)vinyl)-1 - methylquinolinium iodide, a molecule with a structure similar to (E)-4-(2-(5 - methoxy - 1H - indol - 3 - yl)vinyl)-1 - n - hexylquinolinium iodide, on fixed HeLa cells and cells treated with CCCP. Detailed implementation manners

[0045] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer and more understandable, the following further describes this application in detail in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0046] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural. The character " / " generally represents an "or" relationship between the front and back associated objects.

[0047] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one (item)" or its similar expression below refers to any combination of these items, including any combination of single item (item) or plural items (items). For example, "at least one (item) of a, b, or c", or, "at least one (item) of a, b, and c" can both represent: a, b, c, a - b (that is, a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0048] It should be understood that in various embodiments of this application, the magnitude of the sequence numbers of the above - mentioned processes does not mean the order of execution. Some or all steps can be executed in parallel or successively. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0049] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms of "a", "the" and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0050] In the description of the embodiments of the present application, the weight of the relevant components mentioned not only can refer to the specific content of each component, but also can represent the proportional relationship of the weights between the components. Therefore, as long as the content of the relevant components in the description of the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the description of the embodiments of the present application. Specifically, the mass described in the description of the embodiments of the present application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0051] Example 1

[0052] This example discloses the synthesis of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-n-hexylquinolinium iodide. The specific steps are as follows:

[0053] First, 200 μL of 4-methylquinoline and 236 μL of iodohexane are dissolved in ethanol, and the mixture is heated and stirred under reflux at 85 °C for three days. Then, an ethanol solution containing 0.263 g of 5-methoxy-3-formylindole is added. After stirring evenly, 4 to 5 drops of piperidine are added, and the solution gradually turns red. After refluxing for one day, the excess solvent is distilled off, and the product is purified by column chromatography using dichloromethane / methanol as the eluent to obtain a purple-red powder with a yield of about 26%.

[0054] 1 H NMR (400 MHz, DMSO-d 6 ) δ (ppm): 12.09 (s, 1H), 9.12 (d, J = 4.0 Hz, 1H), 8.96 (d, J = 8.0 Hz, 1H), 8.62 (d, J = 16.0 Hz, 1H), 8.44 (m, 3H), 8.18 (t, J = 8.0 Hz, 1H), 7.98 (m, 2H), 7.69 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 6.91 (dd, J = 4.0, 8.0 Hz, 1H), 4.85 (t, J = 8.0 Hz, 2H), 3.90 (s, 3H), 1.92 (m, 2H), 1.32 (m, 6H), 0.86 (t, J = 8.0 Hz, 3H). 13 C NMR (400 MHz, DMSO-d 6), δ (ppm): 155.72, 154.17, 146.12, 139.13, 138.29, 135.13, 132.63, 132.44, 128.80, 127.24, 126.96, 126.14, 119.25, 115.07, 113.92, 113.79, 112.96, 112.82, 102.75, 56.17, 56.11, 31.17, 29.67, 25.97, 22.45, 14.32. HRMS: calculated 385.23, found 385.23.

[0055] In order to develop more compounds with similar functions, in the present invention, the molecular parent nucleus skeleton is kept unchanged, and R 1 may also be arbitrarily selected from any one of C1-C4 alkyl groups, and R 2 may also be arbitrarily selected from any one of hydrogen, C1-C4 alkyl groups, and C1-C4 alkoxy groups. X is selected from halogen atoms, BF 4 and ClO 4 any one of them. According to the research of the inventors, the function of targeting mitochondria without depending on the mitochondrial membrane potential in the present invention is determined by the conjugated organic cation group and has nothing to do with the anion X - ; R 1 is arbitrarily selected from any one of hydrogen or C1-C4 alkyl groups; R 2 is arbitrarily selected from any one of hydrogen, C1-C4 alkyl groups, and C1-C4 alkoxy groups. Changes within this range do not affect the function of the molecule; however, the length of the carbon chain connected to quinoline N has a greater impact on the targeting property, protein binding effect, and molecular aggregation state of the molecule. For example, indolylmethyl quinoline salt binds to RNA in fixed cells, while the indolylhexyl quinoline salt of the present invention localizes in mitochondria.

[0056] Test Example 1

[0057] Culture of HeLa and HEK293 cells:

[0058] The cancer cells HeLa and normal cells HEK293 were cultured in a medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a saturated humidity incubator at 37 °C and 5% CO 2 and subcultured once every 2 - 3 days.

[0059] When the cells grew to the logarithmic phase, they were transferred to a confocal dish for culture: The cells grown in a T25 cell culture flask were first washed with PBS, then digested with 1 mL of trypsin for 1 - 2 minutes (0.25% trypsin for HeLa and 0.025% trypsin for HEK293), the trypsin was removed, fresh medium was added and the cells were pipetted evenly and counted. The cell density was controlled by the addition amount of the medium to make the final cell concentration 1x10 5, and then inoculated into a confocal bottom culture dish and placed in 5% CO 2 Incubate in an incubator, and wait until the cell growth reaches a coverage rate of about 70% for cell experiments.

[0060] Test Example 2

[0061] Staining observation of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide on fixed HeLa cells: After washing the HeLa cells in the confocal dish prepared in Test Example 1 twice with PBS, the following staining steps were then carried out: (1) Incubate with 0.5 μM commercial mitochondrial deep red fluorescent probe solution for 30 min and wash with PBS; (2) Fix the cells with 4% paraformaldehyde solution for 20 min and wash with PBS; (3) Incubate with 1 μM (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide fluorescent probe solution for 30 min and wash with PBS; (4) Incubate with 0.5 μM commercial mitochondrial green fluorescent probe solution for 30 min and wash with PBS. The stained cell samples were observed for multi-channel fluorescence co-localization using a confocal fluorescence microscope.

[0062] The results are as Figure 1 shown, Figure 1 (A) is the red fluorescence image of the probe molecule of the present invention, Figure 1 (B) is the fluorescence image of the commercial mitochondrial deep red fluorescent probe, Figure 1 (C) is the fluorescence image of the commercial mitochondrial green fluorescent probe, Figure 1 (D) is Figure 1 (A) and Figure 1 (B) superimposed image, Figure 1 (E) is Figure 1 (C) and Figure 1 (B) superimposed image. Figure 1 (A) and 1(B) have good overlap, indicating that the fluorescence of the probe molecule of the present invention is completely distributed in the mitochondria; while Figure 1 (C) and 1(B) do not have good fluorescence overlap, indicating that the commercial mitochondrial fluorescent probe cannot be applied to fixed cells. This result proves that (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide, independent of the mitochondrial membrane potential, can be used for mitochondrial staining and fluorescence imaging of fixed cancer cells.

[0063] Test Example 3

[0064] Staining observation of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide on HeLa cells treated with CCCP:

[0065] After washing the HeLa cells in the confocal dish prepared in Test Example 1 twice with PBS, the following treatments and stainings were then carried out: (1) incubating with a 0.5 μM commercial mitochondrial deep red fluorescent probe solution for 30 min, and washing with PBS; (2) incubating with a 20 μM mitochondrial proton binder CCCP solution for 3 h, and washing with PBS; (3) incubating with a 1 μM (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-n-hexylquinolinium iodide fluorescent probe solution for 30 min, and washing with PBS; (4) incubating with a 0.5 μM commercial mitochondrial green fluorescent probe solution for 30 min, and washing with PBS. The stained cell samples were observed for multi-channel fluorescence co-localization using a confocal fluorescence microscope.

[0066] The results are as Figure 2 shown, Figure 2 (A) is the red fluorescence image of the probe molecule of the present invention, Figure 2 (B) is the fluorescence image of the commercial mitochondrial deep red fluorescent probe, Figure 2 (C) is the fluorescence image of the commercial mitochondrial green fluorescent probe, Figure 2 (D) is Figure 2 (A) and Figure 2 (B) superimposed image, Figure 2 (E) is Figure 2 (C) and Figure 2 (B) superimposed image. Figure 2 (A) and 2(B) have good overlap, indicating that the fluorescence of the probe molecule of the present invention is completely distributed in mitochondria; while Figure 2 (C) and 2(B) do not have good fluorescence overlap, indicating that the commercial mitochondrial fluorescent probe cannot be used for staining the mitochondria of cells after the loss of mitochondrial membrane potential. This result proves that (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-n-hexylquinolinium iodide can be used for mitochondrial staining and fluorescence imaging after the loss of mitochondrial membrane potential in living cells.

[0067] Test Example 4

[0068] Staining observation of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-n-hexylquinolinium iodide on HEK293 cells

[0069] After washing the HEK293 cells in the confocal dish prepared in Test Example 1 twice with PBS, the following treatments and stainings were then carried out: (1) incubating with 0.5 μM commercial mitochondrial deep red fluorescent probe solution for 30 min, and washing with PBS; (2) fixing the cells with 4% paraformaldehyde solution for 20 min, and washing with PBS; (3) incubating with 1 μM (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide fluorescent probe solution for 30 min, and washing with PBS. The stained cell samples were observed for multi-channel fluorescence co-localization using a confocal fluorescence microscope.

[0070] The results are as Figure 3 shown, Figure 3 (A) is the red fluorescence image of the probe molecule of the present invention, Figure 3 (B) is the fluorescence image of the commercial mitochondrial deep red fluorescent probe, Figure 3 (C) is Figure 3 (A) and Figure 3 (B) superimposed image. Figure 1 (A) and 1(B) have good overlap, indicating that the fluorescence of the probe molecule of the present invention is completely distributed in mitochondria, proving that (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide can be used for mitochondrial staining and fluorescence imaging of fixed normal cells with mitochondrial membrane potential loss.

[0071] Test Example 5

[0072] Staining observation of the structurally similar control molecule (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-methylquinolinium iodide on HeLa cells with mitochondrial membrane potential loss

[0073] After washing the two groups of HeLa cells in the confocal dish prepared in Test Example 1 twice with PBS, one group was subjected to the following treatments and stainings: (1) fixing the cells with 4% paraformaldehyde solution for 20 min, and washing with PBS; (2) incubating with 10 μM (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-methylquinolinium iodide solution for 30 min, and washing with PBS. The other group was subjected to the following treatments and stainings: (1) incubating with 10 μM (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-methylquinolinium iodide solution for 30 min, and washing with PBS; (2) incubating and preserving with 20 μM mitochondrial protonophore CCCP solution. The two groups of cell samples were observed using a confocal fluorescence microscope.

[0074] The results are as Figure 4 shown, Figure 4(A) Fluorescence photograph of fixed cells stained with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-methylquinolinium iodide Figure 4 (B) Fluorescence photograph of CCCP-treated cells stained with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-methylquinolinium iodide. As can be seen from the figure, the fluorescence of the two groups of cells is distributed in the cytoplasm and nucleoli, which is significantly different from the distribution of mitochondria. It shows that (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-methylquinolinium iodide with a molecular structure similar to the probe of the present invention cannot be used for mitochondrial fluorescence imaging of cells with loss of mitochondrial membrane potential.

[0075] In summary, the present application provides a mitochondrial fluorescence probe that does not depend on mitochondrial membrane potential and its application in mitochondrial imaging observation for non-diagnostic and therapeutic methods. Compared with other mitochondrial fluorescence probes, the unique feature of the probe of the present invention is that it does not depend on a high mitochondrial membrane potential and can target and image the morphology, quantity, and distribution of mitochondria in biological samples with reduced or even lost mitochondrial membrane potential, including mitochondria in fixed cells. It does not fluoresce in water / PBS / culture medium (without serum and antibiotics) solution, but emits red fluorescence after binding to mitochondria in cells; at the same time, it has good membrane permeability and good compatibility for counterstaining.

[0076] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A mitochondrial fluorescent probe independent of mitochondrial membrane potential, characterized in that, the mitochondrial fluorescent probe independent of mitochondrial membrane potential is a compound having the structure shown in formula (I), or a pharmaceutically acceptable salt thereof: Among them, the R 1 includes any one of hydrogen or C1-C4 alkyl groups; the R 2 includes any one of hydrogen, C1-C4 alkyl groups, and C1-C4 alkoxy groups; the X includes a halogen atom, BF 4 and ClO 4 any one of them.

2. The mitochondrial fluorescent probe independent of mitochondrial membrane potential according to claim 1, characterized in that, the halogen atom is selected from any one of iodine, bromine and chlorine.

3. The mitochondrial fluorescent probe independent of mitochondrial membrane potential according to claim 1, characterized in that, The R 1 Among them, the alkyl groups of C1 to C4 include any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

4. The mitochondrial fluorescent probe independent of mitochondrial membrane potential according to claim 1, characterized in that, The R 2 Among them, the C1-C4 alkyl groups include any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

5. The mitochondrial fluorescent probe independent of mitochondrial membrane potential according to claim 1, characterized in that, The R 2 Among them, the alkoxy groups of C1 to C4 include any one of methoxy, ethoxy, propoxy and butoxy.

6. The mitochondrial fluorescent probe independent of mitochondrial membrane potential according to any one of claims 1 to 5, characterized in that, the mitochondrial fluorescent probe independent of mitochondrial membrane potential includes (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-hexylquinolinium iodide.

7. A preparation method of the mitochondrial fluorescent probe independent of mitochondrial membrane potential according to any one of claims 1 to 6, characterized in that, the preparation method includes the following steps: S1. 4-Methylquinoline and halohexane are placed in a solvent and refluxed to react to generate 1-hexyl-4-methylquinolinium salt; S2. 1-Hexyl-4-methylquinolinium salt, indole-3-carbaldehyde and a catalyst are mixed, refluxed and reacted, and then impurities are removed; the catalyst includes piperidine.

8. An application of the mitochondrial fluorescent probe independent of mitochondrial membrane potential according to any one of claims 1 to 5 in any one of A1) to A4): A1) Labeling and / or localizing mitochondria for non-disease treatment diagnosis; A2) Monitoring the related life activities of mitochondria for non-disease treatment diagnosis; A3) Preparing a product for labeling and / or localizing mitochondria; A4) Preparing a product for monitoring the related life activities of mitochondria.

9. An application of the mitochondrial fluorescent probe independent of mitochondrial membrane potential according to any one of claims 1 to 5 in preparing a product targeting mitochondria.

10. An application of the mitochondrial fluorescent probe independent of mitochondrial membrane potential according to any one of claims 1 to 5 in preparing a product for mitochondrial imaging.

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